Acanthamoeba myosin II rod is a long alpha-helical dimeric coiled-coil with a flexible hinge containing a helix-breaking proline. Previously, the thermal stability of the complete wild-type rod domain of myosin II (residues 849-1509), a point mutant in which the hinge proline was replaced with alanine (P398A), and a deletion mutant with the whole hinge region deleted (residues 384-408) in 0.6 and 2.2 M KCl, pH 7.5, was determined. Analytical ultracentrifugation was used to characterize the size and shape (frictional ratio) of myosin II wild-type and hinge mutant rods. Sedimentation properties of minifilaments formed by dialysis of the wild-type and point and deletion mutants vs. low ionic strength buffer with 5 mM Mg (II) at pH 7.5 also have been determined. In addition, the flexibility of myosin II rod minifilaments as a function of Mg(II) concentration has been measured by electric birefringence; specifically, the Mg(II)-dependent flexible-to-stiff transitions of native myosin II and the wild-type rod minifilaments were found by Rau to be very similar. In the present studies, mutations of the phosphorylation sites in the C-terminal tail of the myosin II rod (with an amino terminal 694 residue flag) have substituted 3 Asp for 3 Ser [S1489D, S1494D, S1499D] (78,615 MW), 3 Ala for 3 Ser [S1489A, S1494A, S1499A] (78,483 MW), 1 Asp for Ser [S1489D] (78,559 MW); wild-type rod has 78,531 MW. The wild-type and mutant rods will be used by B. Kunnummal (LCB) to make cofilaments with native myosin II to test if the actin-activated ATPase activity of myosin II heads is inhibited by copolymerized rods containing the Ser-to-Asp substitutions (made to mimic phosphorylation at these sites). If so, the Ser-to-Ala substituted rods in cofilaments with myosin II will act as controls by showing no effect on the head ATPase activity. What we have determined in the Section on Protein Chemistry, LB, is that the thermal stabilities of the wild-type rod dimer and the three phosphorylation- site mutant rods are very similar, using equilibrium circular dichroism measurements at 222 nm and a differential scanning calorimeter (VP-DSC) at a slow scan rate of 4.89 C per hour. Sedimentation velocity experiments demonstrated that all rod preparations in 0.6 M KCl at pH 7.5 are dimeric with ellipsoidal axial ratios of about 45. - myosin II rod, hinge mutants, phosphorylation site mutants, coiled-coil, minifilaments, Acanthamoeba, thermal stability, sedimentation